ABSTRACT Chemotherapy-induced peripheral neuropathy (CIPN) is a common (prevalence 30?70%) and potentially dose- limiting side effect of many cancer chemotherapy drug treatment regimens. Clinically, CIPN presents with pain that is burning, shooting or electric-shock-like. The increase in prevalence of cancer coupled with an increase in the cancer survival rates due to chemotherapy regimens is transforming cancer pain into a large, unmet medical problem. Neurotoxic chemotherapeutic agents (e.g., antimicrotubule agents like paclitaxel) may cause structural damage to peripheral nerves, resulting in aberrant somatosensory processing in the peripheral and/or central nervous system. Dorsal root ganglia (DRG) sensory neurons as well as neuronal cells in the spinal cord are the preferential sites in which chemotherapy induced neurotoxicity occurs. Pathogenesis is complex but includes alterations in ion channels. Paclitaxel (Taxol) increases T-type (Cav3.2) voltage-gated Ca2+ currents in rat dorsal root ganglion (DRG) neurons; these neurons are responsible for conveying noxious sensory stimuli, suggesting these channels are important mediators of specific sensory abnormalities associated with CIPN. Given the roles of these channels in regulating afferent fiber excitability and synaptic function in the spinal dorsal horn and their dynamic regulation during pain states, blocking or depleting Cav3.2 channels in these tissues should mediate analgesic effects. In the past 5 years, considerable effort has been applied towards identifying novel classes of T-type calcium channel blockers. This proposal aims to develop potent, orally available, and selective Cav3.2 channel antagonists, building on the structure of a natural product ? betulinic acid (BA) ? identified by the laboratory of Dr. Leslie Gunatilaka (Director, Natural Products Center, University of Arizona (UA)) and characterized by the PI Dr. Rajesh Khanna (UA) to be Cav3.2-selective and antinociceptive in CIPN. For this work, we have partnered with Regulonix, LLC for characterizing select Cav3.2-targeted compounds and their analogs in in vitro and in vivo efficacy assays as well as early ADME and PK optimization. The work proposed here is the first step in developing non-opioid pain treatments for CIPN that also curb opioid misuse and addiction. We anticipate success against paclitaxel-induced chronic pain to translate into other chronic pain types as well, but CIPN provides focus for early stage proof-of-concept. Regulonix?s specific aims are: (1) Design and synthesis (Dr. L. Gunatilaka) of BA analogues and elucidation of Cav3.2 specificity and biophysical properties of select BA analogs to gain mechanistic and safety information and to document the unique pathway for function in neurons; (2) Profile BA analogues for their in vitro cellular cytotoxicity, early ADME and pharmacokinetic properties, and screening for off-target effects on GPCRs, ion channels and alternative known pain targets, including opioid receptors; and (3) Characterize the best two BA analogs, from Aim 2, for preclinical studies using a neuropathic pain model (paclitaxel) to provide information about oral efficacy, safety, and opioid- sparing. Upon completion, we expect to have a validated BA analog and several worthy backup compounds.